Cutting Edge: NOX2 NADPH Oxidase Controls Infection by an Intracellular Bacterial Pathogen through Limiting the Type 1 IFN Response.

The NOX2 NADPH oxidase (NOX2) produces reactive oxygen species to kill phagosome-confined bacteria. However, we previously showed that Listeria monocytogenes is able to avoid the NOX2 activity in phagosomes and escape to the cytosol. Thus, despite the established role of NOX2 limiting L. monocytogenes infection in mice, the underlying mechanisms of this antibacterial activity remain unclear. In this article, we report that NOX2 controls systemic L. monocytogenes spread through modulation of the type I IFN response, which is known to be exploited by L. monocytogenes during infection. NOX2 deficiency results in increased expression of IFN-stimulated genes in response to type I IFN and leads to 1) promotion of cell-to-cell spread by L. monocytogenes, 2) defective leukocyte recruitment to infection foci, and 3) production of anti-inflammatory effectors IL-10 and thioredoxin 1. Our findings report a novel antimicrobial role for NOX2 through modulation of type I IFN responses to control bacterial dissemination.

Enrichment of Neutrophils and Monocytes From the Liver Following Either Oral or Intravenous Listeria monocytogenes Infection.

Listeria monocytogenes is a foodborne pathogen that causes serious, often deadly, systemic disease in susceptible individuals such as neonates and the elderly. These facultative intracellular bacteria have been an invaluable tool in immunology research for more than three decades. Intravenous (i.v.) injection is the most commonly used transmission route in mice, but oral models of infection have also been developed in recent years, and these may be more appropriate for many studies. This article includes detailed instructions for use of either foodborne or i.v. inoculation of mice and discusses the rationale for choosing either model. Additionally, a protocol is provided for enrichment of neutrophils and monocytes from the infected liver in a manner that allows for determination of bacterial burden while still providing sufficient cells for use in flow cytometric analysis or in vitro assays. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Foodborne L. monocytogenes infection Support Protocol 1: Preparing L. monocytogenes for foodborne infection Basic Protocol 2: Intravenous L. monocytogenes infection Support Protocol 2: Preparing L. monocytogenes for intravenous infection Basic Protocol 3: Enrichment of non-parenchymal cells from the infected liver.

Experimental listeriosis: A study of purinergic and cholinergic inflammatory pathway.

The cholinergic, purinergic and oxidative stress systems were related to nervous system damage in some pathologies, as well as being involved in pro-inflammatory and anti-inflammatory pathways. The objective was to investigate changes in purinergic, cholinergic systems and oxidative stress related to the neuropathology of listeriosis. Gerbils were used as experimental models. The animals were divided in two groups: control and infected. The animals were orally infected with 5 × 108 CFU/animal of the pathogenic strain of Listeria monocytogenes. Collected of material was 6 and 12th days post-infection (PI). Infected animals showed moderate mixed inflammatory infiltrates in the liver. The spleen and brain was used for PCR analyses, confirming infection by L. monocytogenes. Increase in number of total leukocytes because of an increase in lymphocytes in infected (P < 0.001). ATP and ADP hydrolysis by NTPDase was lower at 6 and 12th days PI in infected animals than in the control group. ADA (adenosine deaminase) activity was higher on the 6th day PI (P < 0.05) and decreased on the 12th day PI (P < 0.05) in infected animals. AChE (acetylcholinesterase) activity did not differ between groups on the 6th day PI; however, activity decreased in infected group on the 12th day PI (P < 0.05). On the 12th day PI, an increase of oxygen-reactive species levels and lower catalase and superoxide dismutase activities in the infected group was observed, characterizing a situation of cerebral oxidative stress. The inflammatory and oxidative mechanisms are present in listeriosis in asymptomatic animals, and that ectonucleotidases and cholinesterase's are involved in immunomodulation.

Should arboviruses be the only diagnosis hypothesis in pregnant women in endemic areas presenting with a rash?

Abstract Listeria is an unusual pathogen that causes neonatal infection with high morbidity and mortality. We present the case of a premature newborn whose mother had a rash during pregnancy; the newborn had severe early sepsis because of Listeria monocytogenes and histopathologically suggestive findings of the placenta. Obstetricians and neonatologists should suspect listeriosis in cases with compatible epidemiological history, clinical features, and examination findings of the placenta.

Limited Colonization Undermined by Inadequate Early Immune Responses Defines the Dynamics of Decidual Listeriosis.

The bacterial pathogen Listeria monocytogenes causes foodborne systemic disease in pregnant women, which can lead to preterm labor, stillbirth, or severe neonatal disease. Colonization of the maternal decidua appears to be an initial step in the maternal component of the disease as well as bacterial transmission to the placenta and fetus. Host-pathogen interactions in the decidua during this early stage of infection remain poorly understood. Here, we assessed the dynamics of L. monocytogenes infection in primary human decidual organ cultures and in the murine decidua in vivo A high inoculum was necessary to infect both human and mouse deciduas, and the data support the existence of a barrier to initial colonization of the murine decidua. If successful, however, colonization in both species was followed by significant bacterial expansion associated with an inability of the decidua to mount appropriate innate cellular immune responses. The innate immune deficits included the failure of bacterial foci to attract macrophages and NK cells, cell types known to be important for early defenses against L. monocytogenes in the spleen, as well as a decrease in the tissue density of inflammatory Ly6Chi monocytes in vivo These results suggest that the infectivity of the decidua is not the result of an enhanced recruitment of L. monocytogenes to the gestational uterus but rather is due to compromised local innate cellular immune responses.

The class II phosphatidylinositol 3-phosphate kinase PIK3C2A promotes Shigella flexneri dissemination through formation of vacuole-like protrusions.

Intracellular pathogens such as Shigella flexneri and Listeria monocytogenes achieve dissemination in the intestinal epithelium by displaying actin-based motility in the cytosol of infected cells. As they reach the cell periphery, motile bacteria form plasma membrane protrusions that resolve into vacuoles in adjacent cells, through a poorly understood mechanism. Here, we report on the role of the class II phosphatidylinositol 3-phosphate kinase PIK3C2A in S. flexneri dissemination. Time-lapse microscopy revealed that PIK3C2A was required for the resolution of protrusions into vacuoles through the formation of an intermediate membrane-bound compartment that we refer to as a vacuole-like protrusion (VLP). Genetic rescue of PIK3C2A depletion with RNA interference (RNAi)-resistant cDNA constructs demonstrated that VLP formation required the activity of PIK3C2A in primary infected cells. PIK3C2A expression was required for production of phosphatidylinositol 3-phosphate [PtdIns(3)P] at the plasma membrane surrounding protrusions. PtdIns(3)P production was not observed in the protrusions formed by L. monocytogenes, whose dissemination did not rely on PIK3C2A. PIK3C2A-mediated PtdIns(3)P production in S. flexneri protrusions was regulated by host cell tyrosine kinase signaling and relied on the integrity of the S. flexneri type 3 secretion system (T3SS). We suggest a model of S. flexneri dissemination in which the formation of VLPs is mediated by the PIK3C2A-dependent production of the signaling lipid PtdIns(3)P in the protrusion membrane, which relies on the T3SS-dependent activation of tyrosine kinase signaling in protrusions.

Distribución de serotipos de Listeria monocytogenes aislados de alimentos, Colombia, 2000-2009 / Distribution of Listeria monocytogenes serotypes isolated from foods, Colombia, 2000-2009

Introducción. Listeria monocytogenes es un patógeno facultativo intracelular, oportunista, causante de graves infecciones en humanos, como meningitis, encefalitis y bacteriemias; también, es causa de abortos. Los alimentos actúan como medio de transporte para infectar al huésped. La serotipificación ha discriminado trece serotipos: 1/2a,1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 7. El 4b es causante de la mayoría de listeriosis en el mundo. Objetivo. Determinar la frecuencia en Colombia de los serotipos de L. monocytogenes aislados de alimentos, durante los años 2000-2009. Materiales y métodos. Se trata de un estudio descriptivo y retrospectivo. Se analizaron 1.599 aislamientos, los cuales fueron confirmados como L. monocytogenes y otras especies de Listeria, con pruebas bioquímicas recomendadas por la Food and Drug Administration (Estados unidos) y utilización del sistema bioquímico api Listeria Biomérieux,serotipificadas con la metodología de Seeliger y Höhne. Resultados. De los 1.599 aislamientos, 1.424 fueron confirmados como L. monocytogenes. Los serotipos encontrados fueron: 1/2a con 135 (9,5 %); 1/2b, 154 (10,8 %); 1/2c, 68 (4,8 %); 3a, 4 (0,3 %); 3b, 29 (2,0 %); 3c,2 (0,1 %); 4a, 44 (3,1 %); 4b, 820 (57,6 %); 4c, 6 (0,4 %); 4d- 4e, 140 (9,8 %); 4e, 17 (1,2 %); 7, 2 (0,1 %); y tres no serotipificables, (0,2 %). Los aislamientos procedían principalmente de Bogotá, 1.035 (73 %); de Antioquia, 199 (14 %); de Nariño, 109 (8 %); del Valle del Cauca, 50 (3,5 %), y de otros departamentos, 33 (2,3 %). Conclusión. En los aislamientos analizados, 1.424 (89 %) correspondieron a L. monocytogenes, presentando una buena calidad en el aislamiento e identificación; la mayoría de estos aislamientos pertenecían al serotipo 4b, 820 (57,6 %), serotipo muy virulento. Se recomienda la vigilancia obligatoria de este microorganismo.

Introduction. Listeria monocytogenes is an intracellular, opportunistic pathogen which can cause severe infections such as meningitis, encephalitis and bacteremia. It can also cause abortions in human beings. Foods are the vehicle for infection of the host. Serotypification has discriminated 13 serotypes: 1/2a,1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 7. 4b is the cause of the majority of cases of listeriosis in the world. Objective. The frequency of serotypes of L. monocytogenes was determined in bacteria isolated from foods in Colombia. Materials and methods. The study is descriptive and retrospective. Over a 10-year period, 2000-2009, 1,599 isolates were examined. All were confirmed as Listeria monocytogenes and other strains of Listeria, using biochemical tests recommended by the Food and Drug Administration (USA) and API Listeria and serotyped using the Seeliger and Höhne method. Results. Of the 1,599 isolates, 1,424 were confirmed as L. monocytogenes. Serotypes identified were: 1/2a, 135 (9.5%); 1/2b, 154 (10.8%); 1/2c, 68 (4.8%); 3a, 4 (0.3%); 3b, 29 (2.0%); 3c, 2 (0.1%); 4a, 44 (3.1%); 4b, 820 (57.6%); 4c, 6 (0.4%); 4d- 4e, 140 (9.8%); 4e, 17 (1.2%); 7, 2 (0.1%); not susceptible of serotypification, three cases, (0.2%). Isolates came mainly from the Capital District of Bogotá, 1,035 (73%); from Antioquia 199 (14%), from Nariño, 109 (8%); from Valle del Cauca 50 (3,5%) and from other provinces 33 (2.3%). Conclusion. Of the analyzed isolates, 1,424 (89%) belonged to L. monocytogenes, showing a good quality in isolation and identification. Most of these isolates belonged to serotype 4b, 820 (57.6%), a highly virulent serotype. Obligatory surveillance of this microorganism is recommended.

A small-molecule screen identifies the antipsychotic drug pimozide as an inhibitor of Listeria monocytogenes infection.

We developed a screening procedure to identify small-molecule compounds that altered infection by Listeria monocytogenes to gain insights into bacterial/host cellular processes required for intracellular pathogenesis. A small-molecule library of 480 compounds with known biological functions was screened, and 21 compounds that altered the L. monocytogenes infection of murine bone marrow-derived macrophages (BMM) were identified. The identified compounds affected various cellular functions, such as actin polymerization, kinase/phosphatase activity, calcium signaling, and apoptosis. Pimozide, an FDA-approved drug used to treat severe Tourette's syndrome and schizophrenia, was further examined and shown to decrease the bacterial uptake and vacuole escape of L. monocytogenes in BMM. The inhibitory effect of pimozide on internalization was not specific for L. monocytogenes, as the phagocytosis of other bacterial species (Bacillus subtilis, Salmonella enterica serovar Typhimurium, and Escherichia coli K12) was significantly inhibited in the presence of pimozide. The invasion and cell-to-cell spread of L. monocytogenes during the infection of nonprofessional phagocytic cells also was decreased by pimozide treatment. Although pimozide has been reported to be an antagonist of mammalian cell calcium channels, the infection of BMM in a calcium-free medium did not relieve the inhibitory effects of pimozide on L. monocytogenes infection. Our results provide a generalizable screening approach for identifying small-molecule compounds that affect cellular pathways that are required for intracellular bacterial pathogenesis. We also have identified pimozide, a clinically approved antipsychotic drug, as a compound that may be suitable for further development as a therapeutic for intracellular bacterial infections.

Conjugated action of two species-specific invasion proteins for fetoplacental listeriosis.

The ability to cross host barriers is an essential virulence determinant of invasive microbial pathogens. Listeria monocytogenes is a model microorganism that crosses human intestinal and placental barriers, and causes severe maternofetal infections by an unknown mechanism. Several studies have helped to characterize the bacterial invasion proteins InlA and InlB. However, their respective species specificity has complicated investigations on their in vivo role. Here we describe two novel and complementary animal models for human listeriosis: the gerbil, a natural host for L. monocytogenes, and a knock-in mouse line ubiquitously expressing humanized E-cadherin. Using these two models, we uncover the essential and interdependent roles of InlA and InlB in fetoplacental listeriosis, and thereby decipher the molecular mechanism underlying the ability of a microbe to target and cross the placental barrier.

Glycine betaine improves Listeria monocytogenes tolerance to desiccation on parsley leaves independent of the osmolyte transporters BetL, Gbu and OpuC.

AIMS: To investigate the effect of glycine betaine (GB) on the survival of Listeria monocytogenes on leaf surfaces under low relative humidity (RH). METHODS AND RESULTS: The addition of GB (> or = 25 mmol l(-1)) improved the survival of L. monocytogenes under low RH on parsley leaves, thus suggesting that GB can improve the tolerance of L. monocytogenes to desiccation. Ten times less GB was needed to improve L. monocytogenes survival under low RH on nonbiological surfaces compared with parsley leaves, suggesting that, on the leaf surface, L. monocytogenes may have to compete for the available GB with autochthonous bacteria and/or the plant itself. Wild type and mutants carrying deletions in the three GB uptake systems, BetL, Gbu and OpuC, behaved similarly with and without added GB on parsley leaves (P > 0.05). In addition, preaccumulation of GB, triggered by osmotic stress prior to inoculation, failed to improve survival under low RH compared with osmotic stress without GB accumulation. CONCLUSIONS: Exogenous GB had a protective effect on L. monocytogenes cells from desiccation during survival on parsley leaves. This effect was independent of intracellular GB accumulation by the known uptake systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Presence of GB could improve the survival of L. monocytogenes to desiccation on leaf surfaces and nonbiological surfaces.

Detection of multiple virulence-associated genes in Listeria monocytogenes isolated from bovine mastitis cases.

Clinical samples (n=725) were collected from bovines (n=243) which were positive for mastitis using the California mastitis test (CMT) and somatic cell count (SCC). The clinical samples comprising blood (n=239), milk (n=243), and faecal swabs (n=243) were examined for the presence of pathogenic Listeria spp. Isolation of the pathogen was done using selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar. Confirmation of the isolates was based on biochemical tests and Christie, Atkins, Munch-Petersen (CAMP) test followed by pathogenicity testing. Pathogenicity of the isolates was tested by phosphatidylinositol-specific phospholipase C (PI-PLC) assay as well as in vivo tests namely, chick embryo and mice inoculation tests. The isolates were subjected to PCR assay for five virulence-associated genes, plcA, prfA, hlyA, actA and iap. Listeria spp. were isolated from 12 (1.66%) samples. Of these 4 (0.55%) and 1 (0.14%) were confirmed as Listeria monocytogenes and Listeria ivanovii, respectively. L. monocytogenes and L. ivanovii were recovered from milk samples (2) and faecal (3) of mastitic cattle (3) and buffaloes (2). L. monocytogenes recovered from the milk of mastitic cattle and L. ivanovii from the faecal swab of buffalo turned out to be pathogenic. However, the remaining three hemolytic isolates exhibiting positive CAMP test turned out to be negative in PI-PLC assay, chick embryo and mice inoculation. L. monocytogenes and L. ivanovii isolates characterized as pathogenic by PI-PLC assay and in vivo pathogenicity tests were found to possess all the five virulence-associated genes and three genes, plcA, prfA and actA respectively. The remaining three hemolytic but non-pathogenic L. monocytogenes isolates were negative for plcA by PCR. It seems that the plcA gene and its expression (in the PI-PLC assay) have an important role as virulence determinants in pathogenic Listeria spp. In conclusion, the PI-PLC assay and virulence genes targeted PCR (plcA, prfA and hlyA genes for L. monocytogenes and plcA, prfA and actA genes for L. ivanovii) hold a good promise as rapid and reliable in vitro alternatives to in vivo pathogenicity tests.

Modification of a virulence-associated phenotype after growth of Listeria monocytogenes on food.

AIM: To assess the effect of different foods, which have been implicated or not in cases of listeriosis, on the in vitro virulence-associated phenotype level of different Listeria monocytogenes strains. METHODS AND RESULTS: The virulence-associated phenotype level of L. monocytogenes was studied with the in vitro cell test based on a plaque-forming assay with a human adenocarcinoma cell line (HT-29) monolayer. Three strains of L. monocytogenes were grown in preparations (homogenate, 1-mum filtrate or 0.2-mum filtrate) of different food extracts ['rillettes' (potted minced pork), milk, raw salmon and cold-smoked salmon] or in a control medium, brain heart infusion (BHI). The bacterial suspensions grown in food extracts or in BHI at 37 degrees C were diluted with their growth medium (food extract or BHI) or with minimum essential medium before seeding on confluent HT-29 cell monolayers. Filtration of food extracts had no significant effect on the plaque numbers formed by the bacteria. A significant decrease in the plaque numbers was noted for the three strains when they grew in the rillettes extracts, compared with the other food extracts and BHI. The levels of in vitro virulence-associated phenotype of the strains after growth in the rillettes extract were similar to or lower than that of the hypovirulent internal reference strain L. monocytogenes 442. After growth in milk and cold-smoked salmon, the impact on virulence-associated phenotype depended on the strain. In contrast, plaque-forming assay indicated increased virulence-associated phenotype when the strains were switched from a nutrient-rich medium (food extract or BHI) to a minimum essential medium. CONCLUSIONS: In vitro virulence-associated phenotype level of the studied strains grown in BHI or cold-smoked salmon was the same as the control virulent strain EGD. In contrast, the nutrients present in rillettes may therefore substantially reduce the number of plaques but not the growth of L. monocytogenes. The utilization of minimum essential medium as diluent attenuates changes the effect of the food extract on virulence-associated phenotype in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: In the experimental design of this study, we showed that the nature of the food could affect the in vitro virulence-associated phenotype level of L. monocytogenes.

Listeriosis in the pregnant guinea pig: a model of vertical transmission.

Feto-placental infections represent a major cause of pregnancy complications, and yet the underlying molecular and cellular mechanisms of vertical transmission are poorly understood. Listeria monocytogenes, a facultative intracellular pathogen, is one of a group of pathogens that are known to cause feto-placental infections in humans and other mammals. The purpose of this study was to evaluate possible mechanisms of vertical transmission of L. monocytogenes. Humans and guinea pigs have a hemochorial placenta, where a single layer of fetally derived trophoblasts separates maternal from fetal circulation. We characterized L. monocytogenes infection of the feto-placental unit in a pregnant guinea pig model and in primary human trophoblasts and trophoblast-derived cell lines. The clinical manifestations of listeriosis in the pregnant guinea pigs and the tropism of L. monocytogenes to the guinea pig placenta resembled those in humans. Trophoblast cell culture systems were permissive for listerial growth and cell-to-cell spread and revealed that L. monocytogenes deficient in internalin A, a virulence factor that mediates invasion of nonphagocytic cells, was 100-fold defective in invasion. However, crossing of the feto-placental barrier in the guinea pig model was independent of internalin A, suggesting a negligible role for internalin-mediated direct invasion of trophoblasts in vivo. Further understanding of vertical transmission of L. monocytogenes will help in designing more effective means of treatment and disease prevention.

Metastatic Listeria monocytogenes infection of the peritoneum in mice with cyclosporine a-induced peritonitis.

Inoculation of mice with Listeria monocytogenes intragastrically or by parenteral routes has not been reported to cause peritonitis. In this study, however, severe listerial peritonitis was induced in mice infected subcutaneously and treated intraperitoneally with cyclosporin A (Cs A) in an oil carrier. In both uninfected and listeria-infected mice, intraperitoneal administration of Cs A consistently produced overexpression of P-selectin in the peritoneal microvasculature and pyogranulomatous inflammation of the peritoneum, suggesting that Cs A causes endothelial damage. We suggest that in listeria-infected mice the non-specific irritant peritonitis induced by the intraperitoneal administration of Cs A results in transfer of listeria-infected phagocytes from the liver and spleen to the peritoneal microvasculature, producing metastatic infection.

Listeria monocytogenes infection in Israel and review of cases worldwide.

Listeria monocytogenes, an uncommon foodborne pathogen, is increasingly recognized as a cause of life-threatening disease. A marked increase in reported cases of listeriosis during 1998 motivated a retrospective nationwide survey of the infection in Israel. From 1995 to 1999, 161 cases were identified; 70 (43%) were perinatal infections, with a fetal mortality rate of 45%. Most (74%) of the 91 nonperinatal infections involved immunocompromised patients with malignancies, chronic liver disease, chronic renal failure, or diabetes mellitus. The common clinical syndromes in these patients were primary bacteremia (47%) and meningitis (28%). The crude case-fatality rate in this group was 38%, with a higher death rate in immunocompromised patients.

Molecular studies on the ecology of Listeria monocytogenes in the smoked fish processing industry.

We have applied molecular approaches, including PCR-based detection strategies and DNA fingerprinting methods, to study the ecology of Listeria monocytogenes in food processing environments. A total of 531 samples, including raw fish, fish during the cold-smoking process, finished product, and environmental samples, were collected from three smoked fish processing facilities during five visits to each facility. A total of 95 (17.9%) of the samples tested positive for L. monocytogenes using a commercial PCR system (BAX for Screening/Listeria monocytogenes), including 57 (27.7%) environmental samples (n = 206), 8 (7.8%) raw material samples (n = 102), 23 (18.1%) samples from fish in various stages of processing(n = 127), and 7 (7.3%) finished product samples (n = 96). L. monocytogenes was isolated from 85 samples (16.0%) using culture methods. Used in conjunction with a 48-h enrichment in Listeria Enrichment Broth, the PCR system had a sensitivity of 91.8% and a specificity of 96.2%. To track the origin and spread of L. monocytogenes, isolates were fingerprinted by automated ribotyping. Fifteen different ribotypes were identified among 85 isolates tested. Ribotyping data established possible contamination patterns, implicating raw materials and the processing environment as potential sources of finished product contamination. Analysis of the distribution of ribotypes revealed that each processing facility had a unique contamination pattern and that specific ribotypes persisted in the environments of two facilities over time (P < or = 0.0006). We conclude that application of molecular approaches can provide critical information on the ecology of different L. monocytogenes strains in food processing environments. This information can be used to develop practical recommendations for improved control of this important food-borne pathogen in the food industry.